Lung inflammation, injury, and proliferative response after repetitive particulate hexavalent chromium exposure

Abstract

Background: Chronic inflammation is implicated in the development of several human cancers, including lung cancer. Certain particulate hexavalent chromium [Cr(VI)] compounds are well-documented human respiratory carcinogens that release genotoxic soluble chromate and are associated with fibrosis, fibrosarcomas, adenocarcinomas, and squamous cell carcinomas of the lung. Despite this, little is known about the pathologic injury and immune responses after repetitive exposure to particulate chromates.

Objectives: In this study we investigated the lung injury, inflammation, proliferation, and survival signaling responses after repetitive exposure to particulate chromate.

Methods: BALB/c mice were repetitively treated with particulate basic zinc chromate or saline using an intranasal exposure regimen. We assessed lungs for Cr(VI)-induced changes by bronchoalveolar lavage, histologic examination, and immunohistochemistry.

Results: Single exposure to Cr(VI) resulted in inflammation of lung tissue that persists for up to 21 days. Repetitive Cr(VI) exposure induced a neutrophilic inflammatory airway response 24 hr after each treatment. Neutrophils were subsequently replaced by increasing numbers of macrophages by 5 days after treatment. Repetitive Cr(VI) exposure induced chronic peribronchial inflammation with alveolar and interstitial pneumonitis dominated by lymphocytes and macrophages. Moreover, chronic toxic mucosal injury was observed and accompanied by increased airway pro-matrix metalloprotease-9. Injury and inflammation correlated with airways becoming immunoreactive for phosphorylation of the survival signaling protein Akt and the proliferation marker Ki-67. We observed a reactive proliferative response in epithelial cells lining airways of chromate-exposed animals.

Conclusions: These data illustrate that repetitive exposure to particulate chromate induces chronic injury and an inflammatory microenvironment that may promote Cr(VI) carcinogenesis.

Keywords: chromium; hexavalent; inflammation; injury; intranasal; lung; proliferation; repair.

Figure 1

Resolution of inflammation in BAL fluid (A–C) and lung sections (D–E) at indicated times after a single exposure to particulate Cr(VI) (50 μL of 0.6 mg/mL basic zinc chromate). (A) Total number of living cells. (B and C) Total number of neutrophils (Gr1+; B) and macrophages (CD11c+; C) determined by FACS analysis. (D and E) Representative bright-field photomicrographs of H&E-stained lung sections (original magnification, 20×) showing (D) airways and (E) alveolar regions. Arrows indicate regions of peribronchial inflammation. (F) Merged fluorescence (460–500 nm) and bright-field microscopy images of Giemsa-stained lung tissue. Bars = 200 μm. *p < 0.05 compared with control.

Figure 2

Effects of repetitive Cr(VI) exposure on airway inflammation. (A) Regimen of repetitive intranasal exposure of mice to 50 μL saline or 0.6 mg/mL basic zinc chromate suspended in saline. Number of neutrophils (Gr1+; B) and macrophages (CD11c+; C) in BAL fluid quantified by FACS analysis for 5–11 chromate-treated animals and 6–7 saline-treated animals, expressed as fold of saline control. (D–I) Bright-field photomicrographs of H&E-stained lung sections obtained from mice 8 days after a single Cr(VI) exposure (D,F,H) or 6 days after the fourth exposure (E,G,I). Boxed areas in D and E (10×) are shown at a higher power in F and G (40×), respectively. (H) Lymphocytes and (I) macrophages in the lung (100×). Macrophages are indicated by asterisks; the arrow points to a binucleated or dividing macrophage. Bars = 200 μm in D and E; 100 μm in F and G; and 30 μm in H and I. *p < 0.05 compared with same-date saline control. **p < 0.05 compared with Cr(VI)-treated animals at day 15.

Figure 3

Repetitive Cr(VI) exposure induced airway injury (A,B), Akt signaling (D–F), and epithelial proliferation (G–J). Representative bright-field photomicrographs of H&E-stained (A,B) or immunohistochemically stained (D–J) lung sections obtained from mice at 24 hr after the fifth exposure to Cr(VI) (E–F and H–J) or saline (A,D,G). In (B) regions of airway with injured epithelium are indicated by asterisks. (C) pro-MMP9 levels measured in BAL fluid by ELISA in 5–8 mice. (D–F) Tissue sections labeled by phospho-specific serine-473 Akt antibody. (G–J) Tissue sections labeled by Ki-67 antibody; arrows indicate cells or regions of cells that are positive for Ki-67. Original magnification, 40×; bars = 100 μm. #p < 0.0001 compared with the same-day saline control, as determined by t-test.

Figure 4

Proliferative response in epithelial cells repetitively exposed to particulate Cr(VI) shown by representative bright-field photomicrographs of H&E-stained lung sections obtained from mice 24 hr after the fifth saline or Cr(VI) exposure. (A,B) Original magnification of 10×; bars = 200 μm. (C,D) Boxed areas in A and B, respectively. Original magnification of 40×; bars = 100 μm.